Anti-CRISPR-mediated Acylation and Bioreversible Esterification for Precision Genome Editing

项目摘要/摘要 集群规则间隔短回文重复(CRISPR)/CRISPR相关(CAS)系统使用 RNA引导的蛋白质(例如，Cas9、Cas12a)切割DNA。Cas9和Cas12a有许多相似之处，并具有 被重新用于人类细胞的基因组编辑，因为它们的可编程性、简单性和 效率，以及在基础研究和医学中的应用。Cas12a通常更高效、更具体 而不是卡斯9。然而，由于Cas蛋白的浓度远远高于DNA靶标的浓度 基因组编辑，长时间的Cas12a活性也会导致脱靶DNA切割，染色体易位， 和遗传毒性。 为Cas12a配备“ON-OFF”开关可以通过缩短Cas12a的 暴露和增加其特异性-DNA切割的靶上与靶外的比率。我们开发了一种 改变CRISPR-Cas抑制蛋白(即，抗CRISPR)的目的以选择性酰化或 带有可拆卸小分子基团的“笼式”Cas12a。我们计划提供小分子和反- CRISPR蛋白通过快速、生物可逆和体内相容的方式进入人类细胞，激活和 分别灭活Cas12a，提高其特异性。在杰出的麻省理工学院专家的帮助下 和博德研究所的设施，我将学习如何使用液相色谱-串联质谱仪 (LC-MS/MS)来评估我们新的Cas12a酰化策略的选择性，以及哺乳动物细胞培养，高 吞吐量共聚焦显微镜、下一代测序和计算分析 抗CRISPR传递和Cas12a基因组编辑特异性。总体而言，本研究将介绍第一个 Cas12a赖氨酸残基的位置选择性和可逆酰化方法及一种新的生物可逆性 用于无迹传递抗CRISPR蛋白的酯化策略，这些蛋白一起将增加基因组- 编辑专一性。

PROJECT SUMMARY/ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems use RNA-guided proteins (e.g., Cas9, Cas12a) to cleave DNA. Cas9 and Cas12a share many similarities and have been repurposed for genome editing in human cells because of their programmability, simplicity, and efficiency, with applications in basic research and medicine. Cas12a is generally more efficient and specific than Cas9. However, since the concentration of Cas protein is much higher than that of the DNA target in genome editing, prolonged Cas12a activity also leads to off-target DNA cleavage, chromosomal translocations, and genotoxicity. Equipping Cas12a with an “on-off” switch can overcome these challenges by shortening Cas12a’s window of exposure and increasing its specificity—the on-target to off-target ratio of DNA cleavage. We developed a novel strategy that repurposes a CRISPR-Cas inhibitor protein (i.e., an anti-CRISPR) to selectively acylate or “cage” Cas12a with a small-molecule-removable group. We propose to deliver small molecules and anti- CRISPR proteins into human cells via fast, bioreversible, and in vivo-compatible means to activate and inactivate Cas12a, respectively, and increase its specificity. With the help of experts at the outstanding MIT and Broad Institute facilities, I will learn how to use liquid chromatography–tandem mass spectrometry (LC–MS/MS) to assess the selectivity of our novel Cas12a acylation strategy, and mammalian cell culture, high throughput confocal microscopy, next-generation sequencing, and computational analysis to assess anti-CRISPR delivery and Cas12a genome-editing specificity. Overall, this research will introduce the first approach for the site-selective and reversible acylation of Cas12a lysine residues and a new bioreversible esterification strategy for the traceless delivery of anti-CRISPR proteins that together will increase genome- editing specificity.